Subscriber access provided by Northern Illinois University
Article
Protective effect of RA on myocardial infarction induced-cardiac fibrosis via AT1R/p38 MAPK pathway signaling and modulation of the ACE2/ACE ratio qiaofeng liu, Tian Jingwei, Yannan Xu, Chunmei Li, Xiangjing Meng, and Fenghua Fu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03001 • Publication Date (Web): 19 Aug 2016 Downloaded from http://pubs.acs.org on August 22, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 21
Journal of Agricultural and Food Chemistry
63x45mm (300 x 300 DPI)
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
1
Protective effect of RA on myocardial infarction induced-cardiac fibrosis via AT1R/p38
2
MAPK pathway signaling and modulation of the ACE2/ACE ratio
3 4
Qiaofeng Liu#, Jingwei Tian#,*, Yanan Xu, Chunmei Li *, Xiangjing Meng, and Fenghua Fu
5
6
7
8
9
10
11
12 13 14 15 16 17 18 19
Abbreviations: HF, heart failure; MI, myocardial infarction; RA, Rosemary acid; ECM, extracellular
20
matrix; Hyp, hydroxyproline; RAS, rein-angiotensin system; ACE, angiotensin-converting enzyme; ACE2,
21
angiotensin-converting enzymes 2; Ang II, angiotensin II; AT1R, angiotensin type 1 receptor; α-SMA, alpha
22
smooth muscle actin; MAPK, mitogen-activated protein kinase; LVSP: left ventricular systolic pressure;
23
LVEDP: left ventricular end-diastolic pressure; HR: heart rate; LV, left ventricular; +dp/dtmax, maximum rats
24
of left ventricular pressure rise; -dp/dtmax, maximum rats of left ventricular pressure fall; HWI, heart weight
25
index; LVWI, left ventricular weight index; CVF, collagen volume fraction; ACEI, ACE inhibitor.
1 ACS Paragon Plus Environment
Page 2 of 21
Page 3 of 21
Journal of Agricultural and Food Chemistry
1
ABSTRACT: Rosmarinic acid (α-o-caffeoyl-3,4-dihydroxyphenyl lactic acid, RA), a major active
2
constituent of Rosmarinus officinalis Linn. (rosemary), having significant anti-inflammatory,
3
anti-apoptotic and anti-oxidant effects. However, the cardioprotection of RA is still not understood.
4
Present study was designed, for the first time, to investigate the cardioprotection of RA on
5
myocardial infarction (MI) induced-cardiac fibrosis and clarify the possible mechanisms. MI was
6
induced in adult rats by left anterior descending coronary artery ligation, and animals were then
7
administered RA (50, 100, 200 mg/kg) by gavage. Compared with the model group, RA treatment
8
ameliorated the changes in left ventricular systolic pressure (LVSP), +dp/dtmax, and −dp/dtmax after 4
9
weeks. This was associated with attenuation of infarct size, collagen volume fraction (CVF),
10
expression of collagen I, collagen III, alpha smooth muscle actin (α-SMA), and hydroxyproline
11
(Hyp) concentrations. RA treatment was also associated with decreased angiotensin-converting
12
enzyme (ACE) expression and increased ACE2 expression, as well as decreased expression of
13
angiotensin type 1 receptor (AT1R) and phospho-p38 mitogen-activated protein kinase (p38
14
MAPK). Thus, RA can protect against cardiac dysfunction and fibrosis following MI, likely due to
15
decreasing ACE expression and increasing ACE2 expression via AT1R/ p38 MAPK pathway.
16 17
KEYWORDS: RA; cardiac dysfunction; cardiac fibrosis; ACE, ACE2, AT1R/p38 MAPK
18
2 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
1
■ INTRODUCTION
2
Myocardial infarction (MI) is the main pathogenic factor underlying heart failure (HF).1 In the
3
post-MI phase, the heart undergoes extensive myocardial remodeling in response to the ischemic
4
injury, leading to thickening or stiffening of regions of the heart, with progressive deterioration of
5
cardiac function, which can progress to HF.2 Cardiac fibrosis plays a major role in cardiac
6
remodeling after MI, and is a predisposing factor for HF. The pathological features of cardiac
7
fibrosis include phenotypic changes in cardiac fibroblasts, excessive proliferation, and deposition of
8
extracellular matrix (ECM) proteins such as collagen types I and III.3
9
The rein-angiotensin system (RAS) is important for the induction and development of cardiac
10
fibrosis after MI.4 Angiotensin-converting enzyme (ACE) and angiotensin-converting enzymes 2
11
(ACE2) control the production of the bioactive angiotensin peptides of the RAS. For example, the
12
balance between ACE and ACE2 (ACE/ACE2) regulates angiotensin II (Ang II) level, and
13
represents a pivotal process in the activation of MI-induced cardiac fibrosis.5 Indeed, an increased
14
ACE/ACE2 ratio is observed in animal models and patients with HF.5 Further, under hypertensive
15
conditions, Ang II increases the ratio of ACE/ACE2 via the angiotensin type 1 receptor (AT1R) and
16
p38 mitogen-activated protein kinase (MAPK) both in vivo and in vitro.6
17
Rosmarinus officinalis Linn. (rosemary), belonging to the family Lamiaceae, is an evergreen
18
aromatic plant with upright stems, dark green leaves, and whitish-blue flowers distributed in the
19
Mediterranean region. 7 Extract of rosemary leave was shown to be a popular herbal products that
20
has been consumed as an anti-oxidant agent and flavoring in cosmetics and food conservation. 8,9 In
21
some countries, rosemary is usually used as a medicinal plant in the modern and traditional
22
medicines for hypertension and diabetes complications treatment. 10-12
23
Rosmarinic acid (Figure 1, α-o-caffeoyl-3,4-dihydroxyphenyl lactic acid, RA), a major active
24
constituent of rosemary, has marked anti-inflammatory, anti-apoptotic, and anti-oxidant effects.13-15
25
Recently, Kim et al. and our previous study reported that RA has antifibrogenic effects in kidney
3 ACS Paragon Plus Environment
Page 4 of 21
Page 5 of 21
Journal of Agricultural and Food Chemistry
1
and liver fibrosis.16-17 The protective effects of RA against cardiac fibrosis have not been reported,
2
although beneficial effects of RA against cardiac cell injury induced by anti-hypoxia and
3
re-oxygenation
4
MAPK expression in vitro and in vivo.20 Thus, in the present study, we examined the hypothesis that
5
long-term oral RA treatment would prevent cardiac dysfunction and cardiac fibrosis following MI
6
in rats, through down-regulating ACE expression and up-regulating ACE2 expression via the
7
AT1R/p38 MAPK pathway.
8
■ MATERIALS AND METHODS
18
and acute ischemia in rats19 were reported. Interestingly, RA also inhibits p38
9
Plant material. RA (Formula: C18H16O8, Figure 1) was supplied by Shandong Engineering
10
Research Center for Nature Drug (Yantai, China) with a purity of > 99.5% as determined by HPLC
11
(Figure S1).
12
Animals. Sprague-Dawley male rats, weighing 260 ± 20 g, were obtained from Shandong
13
Luye Pharmaceutical Co. Ltd. (SCXK Lu 2014 0002), and housed in a specific-pathogen-free grade
14
laboratory at 23 ± 3°C, 40–65% humidity, and on a 12-h light/dark cycle. Before the trial, animals
15
were adapt to the environment for 1 week with free access to food and drinking water. All animals
16
showed normal drinking, eating, and activity, with no adverse reactions. Experimental protocols
17
were approved by the Animal Ethics Committee of Yantai University (IACUU-201500513).
18
Coronary artery ligation surgery. Animals were anesthetized (sodium pentobarbital, 35
19
mg/kg) by intraperitoneal injection (i.p). A left thoracotomy was performed and the heart was
20
ligated around the left anterior descending coronary artery (LAD). The wound was stitched by a 6-0
21
prolene suture after the air was completely vented out. Successful MI was confirmed by apparent
22
ST-segment elevation in electrocardiogram. Sham-operated animals underwent the same surgical
23
procedure without the coronary ligature. All animals received routine intramuscular injections of
24
buprenorphine-HCl (0.2 mg/kg) for analgesia and penicillin (1000 U) to prevent infection.
25
Experimental design. Rats were randomly divided into five groups: (1) sham rats treated
4 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
1
with 0.9% physiologic saline (n = 15), (2) MI rats treated with 0.9% physiologic saline (n = 15), (3)
2
MI rats treated with 50 mg/kg RA (n = 15), (4) MI rats treated with 100 mg/kg RA (n = 15), and (5)
3
MI rats treated with 200 mg/kg RA (n = 15). All rats were treated daily with RA by gastric gavage
4
once a day for 4 weeks from the 1st day after surgery.
5
Assessment of hemodynamic parameters. After 4 weeks, all rats were anesthetized
6
with sodium pentobarbital (35 mg/kg, i.p.) for hemodynamic assessment. A micromanometer-tipped
7
catheter (Model SPR-838; Millar instruments) was introduced via the right carotid artery into the
8
left ventricle to measure left ventricular systolic pressure (LVSP), left ventricular end-diastolic
9
pressure (LVEDP), +dp/dtmax, and −dp/dtmax of the left ventricle.
10
Assessment of heart weight index and left ventricular weight index. After
11
hemodynamic measurements, blood samples were taking from the abdominal aorta, centrifuged,
12
and the supernatant was stored at −20°C. The heart and the left ventricle (LV) were isolated, washed,
13
and weighed. Relative values (organ weight/body weight) of the heart weight index (HWI, mg/g)
14
and the left ventricular weight index (LVWI, mg/g) were determined.
15
Assessment of infarct size and cardiac fibrosis. The left ventricles of the tissue below
16
the ligature line (3–5 mm thick) were then fixed with 4% paraformaldehyde, dehydrated, and
17
embedded in paraffin. To assay the degree of cardiac fibrosis, the collagenous fibrotic area fraction
18
was assessed using the Masson’s trichrome staining kit (Maixin Bio-Technology Co., Ltd, Fuzhou,
19
China) in paraffin sections (4 µm thick). The sections were digitally imaged using a microscope
20
(Olympus DP25) and the collagen volume fraction (CVF) in the peri-infarcted areas of LV was
21
evaluated as the percentage of fibrosis area (blue staining) to the total LV area in an average of five
22
sections in each heart (NIH Image software). The infarct size was calculated as the ratio of total
23
infarct circumference to total LV circumference (NIH Image software).
24
Assessment of hydroxyproline concentrations. The hydroxyproline (Hyp) content in
25
blood serum was assayed using a commercial kit (Nanjing Jiancheng Bioengineering Institute,
5 ACS Paragon Plus Environment
Page 6 of 21
Page 7 of 21
Journal of Agricultural and Food Chemistry
1
Nanjing, China) according to the manufacturers’ instructions. The concentrations of Hyp were
2
expressed as µg/ml.
3
Western blotting. Myocardial tissue was ground in radio-immunoprecipitation assay lysis
4
buffer, and the homogenate was stored on ice for 30 min, centrifuged at 14,000 ×g for 15 min at
5
−4°C, and the supernatant was collected. A BCA kit (Beyotime Institute of Biotechnology, Nanjing,
6
China) was used to determine the protein concentration. The supernatant was diluted in SDS-PAGE
7
loading buffer (5X) and then boiled for 5 min. Protein samples were subjected to SDS-PAGE and
8
transferred to polyvinylidene fluoride membranes. The membranes were blocked in TBST
9
containing 5% skim milk for 2 h at room temperature, and subsequently incubated with the
10
following antibodies: rabbit polyclonal anti-α-SMA antibody (abcam), rabbit polyclonal
11
anti-collagen I antibody (abcam) and anti-collagen III antibody (abcam), anti-Ang II antibody
12
(abcam), anti-p38 antibody (abcam) and anti-phospho-p38 antibody (abcam), anti-AT1R antibody
13
(abcam), rabbit polyclonal anti-ACE antibody (Santa Cruz) and anti-ACE2 antibody (Santa Cruz),
14
and mouse monoclonal anti-GADPH antibody (Beyotime) for overnight at -4℃. The membranes
15
were then washed three times with TBST, probed with appropriate secondary antibodies for 1.5 h at
16
room temperature, and proteins detected by chemiluminescence.
17
Statistical analysis. All data were reported as mean ± SD. Inter-group differences were
18
analyzed with one-way analysis of variance (ANOVA) followed by Dunnett's test. The analyses
19
were performed using Graph Pad Prism Software, V5.0. Differences were considered statistically
20
significant at a value of P < 0.05.
21 22
■ RESULTS
23
Effects on cardiac function. Hemodynamic outcomes obtained at 4 weeks after MI are
24
shown in Table 1. After MI, there was a decrease in diastolic function, as shown by a significant
25
increase in LVEDP (P < 0.05) and decrease in −dp/dtmax (P < 0.01). MI also impaired systolic 6 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
1
cardiac function, with a significant decrease in LVSP (P < 0.01) and +dp/dtmax (P < 0.01). Treatment
2
with RA markedly improved diastolic and systolic cardiac function, as shown by a significant
3
increase in LVSP with 100 mg/kg RA (P < 0.05), and a significant increase in LVSP, +dp/dtmax and
4
−dp/dtmax with 200 mg/kg RA (P < 0.01, P < 0.05), compared with the model group. There were no
5
significant differences in HR between any of the groups.
6
Effects on infarct size, heart weight index and left ventricular weight index. The
7
infarct sizes in the model group and RA groups are shown in Figure 2. There was a significant
8
increase in infarct size in MI rats compared to sham rats, which was obviously decreased with RA
9
treatment compared with the model group (P < 0.05, P < 0.01). As shown in Table 1, HWI and
10
LVWI were markedly higher in the model group compared with the sham group (P < 0.01).
11
However, compared with the model group, there was a trend towards a decrease in HWI and LVWI
12
in the RA groups.
13
Effects on cardiac fibrosis. Also, we assessed the effects of RA on cardiac fibrosis. By
14
Masson’s trichrome stained sections (collagen stains blue), there was a significant increase in the
15
CVF in the model group compared with the sham group (P < 0.01). By contrast, CVF was
16
attenuated in the RA treatment groups (P < 0.01) (Figure 3).
17
Effects on type I and III collagen content, hydroxyproline concentration and
18
α-smooth muscle actin expression. Next, collagen I and collagen III protein content were
19
assessed by Western blotting. There was a significant increase in collagen I and collagen III protein
20
expression in the model group compared with the sham group (P < 0.01 for both), which was
21
markedly reduced with RA treatment (P < 0.01) (Figure 4 A). On the other hand, there was a
22
significant increase in Hyp concentration in the model group compared with the sham group (P
